Department of Nuclear Medicine, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi710032, China.
Departments of Diagnostic Radiology, Surgery, Chemical and Biomolecular Engineering, and Biomedical Engineering, Yong Loo Lin School of Medicine and Faculty of Engineering, National University of Singapore, Singapore, 119074, Singapore.
Bioconjug Chem. 2021 Sep 15;32(9):2108-2116. doi: 10.1021/acs.bioconjchem.1c00392. Epub 2021 Sep 6.
The kidney is the main dose-limiting organ in radioligand therapy (RLT), and there is an urgent need for reducing renal radioactivity accumulation. According to the enzymolysis clearance strategy, the first objective of this study is to test whether enzymolysis efficiency can be improved by introducing a hydrophobic amino acid with a bulkier side chain to the second position of the cleavable sequence, and the second objective is to screen an optimal sequence to minimize the renal uptake. Four exendin 4 (Ex4) peptide analogues with different cleavable sequences were synthesized and labeled with Ga. Both in vitro and in vivo metabolism studies were performed using either the model compounds or the complete probes. The in vitro stabilities of the tracers were evaluated in PBS and mouse serum. The microPET images were acquired in the INS-1 tumor model at different time points, and the radioactivity uptakes of the probes in tumors and kidneys were determined and compared. All the probes were stable in both PBS and mouse serum for at least 1 h. The in vitro cleavage study for both model compounds and intact probes showed enzymolysis efficiency in the following order: MWK > MFK > MVK > MGK. The in vivo metabolism study confirmed that a fragment of Ga-NOTA-Met-OH appeared in both kidney and urine samples for all analogues with MVK, MFK, and MWK sequences. The microPET images showed that the tumor uptakes of all the modified probes were comparable to those of the control, while the kidney uptakes were significantly reduced by inserting the MWK, MFK, or MVK linker. The tumor-to-kidney ratios at 0.5, 1, and 2 h time points showed the following order: Ga-NOTA-MWK-Ex4 > Ga-NOTA-MFK-Ex4 > Ga-NOTA-MVK-Ex4. In this study, based on the enzymolysis clearance strategy and the preference of the enzyme, different sequences were designed and compared both in vitro and in vivo. The results indicated that the larger the steric hindrance of the second hydrophobic amino acid side chain, the more effective the enzymatic hydrolysis, with enzymolysis efficiency in the following order: MWK > MFK > MVK > MGK. MWK appears to be the most effective sequence in reducing renal radioactivity accumulation of exendin 4 peptide derivatives.
肾脏是放射性配体治疗（RLT）中的主要剂量限制器官，因此迫切需要降低肾脏的放射性积累。根据酶解清除策略，本研究的首要目标是测试通过在可切割序列的第二位引入疏水性较大的氨基酸，是否可以提高酶解效率，其次是筛选出一个最佳序列以最小化肾脏摄取。合成了四种不同可切割序列的 Ex4 肽类似物，并与 Ga 标记。使用模型化合物或完整探针进行了体外和体内代谢研究。在 PBS 和鼠血清中评估了示踪剂的体外稳定性。在 INS-1 肿瘤模型的不同时间点采集 microPET 图像，并测定和比较探针在肿瘤和肾脏中的放射性摄取。所有探针在 PBS 和鼠血清中至少 1 小时内均稳定。对于模型化合物和完整探针的体外切割研究表明，酶解效率依次为：MWK＞MFK＞MVK＞MGK。体内代谢研究证实，对于具有 MVK、MFK 和 MWK 序列的所有类似物，均在肾脏和尿液样本中出现了 Ga-NOTA-Met-OH 的片段。microPET 图像显示，所有修饰探针的肿瘤摄取与对照相比均相当，而插入 MWK、MFK 或 MVK 接头后，肾脏摄取明显减少。0.5、1 和 2 小时时间点的肿瘤与肾脏比值依次为：Ga-NOTA-MWK-Ex4＞Ga-NOTA-MFK-Ex4＞Ga-NOTA-MVK-Ex4。在这项研究中，基于酶解清除策略和酶的偏好，设计并比较了不同的序列，包括体外和体内实验。结果表明，第二个疏水性氨基酸侧链的空间位阻越大，酶解越有效，酶解效率依次为：MWK＞MFK＞MVK＞MGK。MWK 似乎是降低 Ex4 肽衍生物肾脏放射性积累的最有效序列。
